SOMATIC HYPERMUTATION

CDR1 CDR2 CDR3 VL J2 gene product V35 gene product Complementary Determining Region = hypervariable region

STRUCTURE OF THE VARIABLE REGION Hypervariable (HVR) or complimentarity determining regions (CDR) HVR3 FR1 FR2 FR3 FR4 HVR1 HVR2 Variability Index 25 75 50 100 Amino acid residue 150 Framework regions (FR)

Szomatikus hipermutáció Variabilitás FR1 CDR1 FR2 CDR2 FR3 CDR3 FR4 100 80 60 40 20 20 40 60 80 100 120 Aminsav szám A különböző specificitású ellenanyagokban található pont mutációk összehasonlítása Wu - Kabat analízissel Mik a következményei az immunválasz során végbemenő mutációknak egy adott epitóp ellen irányuló ellenanyagban? Hogyan befolyásolja az ellenanyag specificitását és affinitását?

LIGHT CHAIN Light chain Disulphide bridges Heavy chain VL CL FR1 FR2 FR3 FR4 CDR1 CDR2 CDR3 Heavy chain VL CL FR1 FR2 FR3 FR4 CDR1 CDR2 CDR3

SOMATIC HYPERMUTATION Hypervariable regions Day 0. Ag Plasma cell clones 1 2 3 4 5 6 7 8 Day 7 PRIMARY immune response AFFINITY MATURATION 9 1011 12 13 14 15 16 Day 14 Day 14. Ag 17 1819 20 21 22 23 24 Day 21 SECONDARY Immune response Hypervariable regions

Hypermutation occurs under the influence of activated T cells Somatic hypermutation leads to affinity maturation Clone 1 Clone 2 Clone 3 Clone 4 Clone 5 Clone 6 Clone 7 Clone 8 Clone 9 Clone 10 CDR1 CDR2 CDR3 Day 6 CDR1 CDR2 CDR3 Day 8 Day 12 Day 18 Deleterious mutation Beneficial mutation Neutral mutation Lower affinity - Not clonally selected Higher affinity - Clonally selected Identical affinity - No influence on clonal selection Hypermutation occurs under the influence of activated T cells Mutations are focussed on ‘hot spots’ (i.e. the CDRs) and are due to double stranded breaks repaired by an error prone DNA repair enzyme.

CDR1 and CDR2 regions are encoded by the V-gene The CDR3 of L-chain is encoded by V and J The CDR3 of H-cain is encoded by V, D and J genes

Hypervariable loops and framework: Summary The framework supports the hypervariable loops The framework forms a compact b barrel/sandwich with a hydrophobic core The hypervariable loops join, and are more flexible than, the b strands The sequences of the hypervariable loops are highly variable amongst antibodies of different specificities The variable sequences of the hypervariable loops influences the shape, hydrophobicity and charge at the tip of the antibody Variable amino acid sequence in the hypervariable loops accounts for the diversity of antigens that can be recognised by a repertoire of antibodies 10

Where and how do all these things take place? B – CELL ACTIVATION Where and how do all these things take place?

B-cell recycling in the absence of antigen (lymph node) B cells in blood T cell area B cell area Efferens lymph

Recirculating B cells are trapped by foreign antigens in lymphoid organs B cells leave blood & enter lymph node via high endothelial venules B cells proliferate rapidly Antigen enters node in afferent lymphatic Y Germinal centre releases B cells that differentiate into plasma cells GERMINAL CENTRE Transient structure of Intense proliferation

Germinal Center Reaction Germinal centers, where B cells proliferate and undergo both isotype switching and somatic hypermutation, form within the B cell follicles in lymphoid organs. The formation of germinal centers starts when dendritic cells displaying antigen on their surface activate antigen-specific CD4 T cells, which proliferate and mature into effector cells capable of activating antigen-specific B cells. Once activated by a CD4 T cell, the B cell proliferates to form a primary focus of antigen specific B cells. B cells from the primary focus migrate to nearby follicles and proliferate. Other B cells from the primary focus persist in the T cell area for a short while, secreting antibody, but eventually die. The B cells that enter the follicle begin to proliferate rapidly. During this time they also undergo somatic mutation, to introduce new variation into the B cell receptor. B cells undergo a process of selection after somatic mutation, when the receptors are tested for their ability to bind antigen. Those that fail to bind, or that fail to compete efficiently against other B cell receptors, die.

„Dating” in the peripheral lymphoid organs

The structure of the germinal centre Somatic hypermutation LZ FDC DZ Somatic hypermutation LZ: light zone DZ: dark zone FDC: follicular dendritic cell

Antigen is bound on the surface of follicular dendritic cells (FDC)  FDC-s bind immune complexes (Ag-Ab )  Ag detectable for 12 months following immunization  A single cell binds various antigens Fig. 9.15. On the surface of FDC-s immune complexes form the so-called iccosomes,that can be released and taken up later by the surrounding germinal center B cells B cells recognize Ag on the surface of FDC This concept is being challenged now, and there has been some backtracking in the current text. It is possible that the FDCs provide persistent Ag to long-lived plasma cells.

T CELL DEPENDENT B CELL ACTIVATION IN LYMPHOID ORGANS IgM IgG IgA IgE

SIGNALING UNITS OF THE B-CELL RECEPTOR Ig-a/CD79a a b Y Ig-b/CD79b Ig domain + CHO ITAM ITAM: YxxL x7 YxxI ITAM: Immunoreceptor Tyrosine-based Activation Motif

Main steps of B-cell signal transduction

CONSEQUENCES OF B-CELL RECEPTOR CROSS LINKING Ag binding, cross-linking of surface Ig Lymphocyte activation Phenotypic/ Functional change Fokozott túlélés Osztódás Ko-stimuláló molekulák kifejeződése Citokin receptorok kifejeződése Kivándorlás a limfoid tüszőből a T sejtes területekre

KINETICS OF LYMPHOCYTE ACTIVATION ANTIGEN SIGNAL1. Nyugvó limfocita G0 sejtosztódás DNA synthesis Effector cell Memory cell Transport Membrane change RNA and protein synthesis Resting lymphocyte G0 Ko-receptor Adhesion molecule Cytokines SIGNAL2. Resting lymphocyte G0 PTK activation RNA synthesis Free Ca++ Protein synthesis Protein phosphorylation DNA synthesis Lymphoblast 0 10sec 1min 5min 1hr 6 hrs 12 hrs 24 hrs

Antigenic determinant C3d THE CO-STIMULATORY ROLE OF CR2 (CD21) COMPLEMENT RECEPTOR IN B – LYMPHOCYTES ANTIGÉN CD21/CR2 CD19 Y TAPA=CD81 B-CELL Enhanced B-cell activation

THE NEURAMIC ACID RECEPTOR CD22 INHIBITS ACTIVATION THROUGH THE A B-CELL RECEPTOR Tissue cells Bacterium Mannose Neuraminic acid B Cell Antigen CD22 Inhibited B cell activation

EFFECTOR FUNCTIONS OF ANTIBODIES INHIBITION Binding of bacteria to epithelial cells Binding of viruses to receptor Binding of bacterial toxins to target cells NEUTRALIZATION Small proportion of antibodies PLASMA CELL OPSONIZATION Binding of antibody increases phagocytosis FcR FcR CR1 Complement C3b COMPLEMENT ACTIVATION Opsonization by C3b PHAGOCYTES ENGULFMENT, DEGRADATION

FEATURES OF THE BINDING SITE SIZE SHAPE HYDROPHOBIC HYDROPHYLIC POSITIVELY CHARGED NEGATIVELY CHARGED ANTIGEN BINDING IS MEDIATED BY NON-COVALENT INTERACTIONS One binding site is able to interact with more than one antigen The strength of interaction (affinity/avidity) varies in a broad range

Growth factors MHC – peptid - TCR Adhesion molecules ANTIBODIES Affinity